1. Technical Field
The invention relates generally to the treatment of muscle loss in a mammal, and more particularly, to the administration of one or more branched chain amino acid(s) (BCAA), a BCAA precursor, a BCAA metabolite, a BCAA-rich protein, a protein manipulated to enrich the BCAA content or any combination thereof in the treatment of such muscle loss. The invention further relates to nutritional formulations suitable for such administration.
2. Background Art
Amino acids are the monomeric building blocks of proteins, which in turn comprise a wide range of biological compounds, including enzymes, antibodies, hormones, transport molecules for ions and small molecules, collagen, and muscle tissues. Amino acids are considered hydrophobic or hydrophilic, based upon their solubility in water, and, more particularly, on the polarities of their side chains. Amino acids having polar side chains are hydrophilic, while amino acids having non-polar side chains are hydrophobic. The solubilities of amino acids, in part, determines the structures of proteins. Hydrophilic amino acids tend to make up the surfaces of proteins while hydrophobic amino acids tend to make up the water-insoluble interior portions of proteins.
Of the common 20 amino acids, nine are considered indispensable (essential) in humans, as the body cannot synthesize them. Rather, these nine amino acids must be obtained through an individual's diet. A deficiency of one or more amino acids can cause a negative nitrogen balance. A negative nitrogen balance, for example, is wherein more nitrogen is excreted than is administered. Such a condition can lead to disruption of enzymatic activity and the loss of muscle mass.
A number of muscle-wasting conditions have been identified for which treatment with amino acid supplements has proved beneficial. For example, cachexia is a severe body wasting condition characterized by marked weight loss, anorexia, asthenia, and anemia. Cachexia is a common feature of a number of illnesses, such as cancer, sepsis, chronic heart failure, rheumatoid arthritis, and acquired immune deficiency syndrome (AIDS). Other muscle wasting diseases and disorders are known, including, for example, sarcopenia, an age-related loss of muscle mass.
Proteolysis-Inducing Factor (PIF)
It has been found that certain tumors may induce cachexia through the production of a 24 kDa glycoprotein called proteolysis-inducing factor (PIF). One proposed mechanism of action of PIF is to decrease protein synthesis; another proposed mechanism of PIF is an activation of protein degradation; a third proposed mechanism is a combination of the aforementioned decrease in protein synthesis and activation of protein degradation. It has been hypothesized that the decreased protein synthesis associated with PIF is the result of PIF's ability to block the translation process of protein synthesis. Another factor, Angiotensin II (Ang II) has shown similar effects and may be involved in the muscle wasting observed in some cases of cachexia.
The original role of PIF in the ubiquitin-proteosome pathway is known. PIF produces an increased release of arachadonic acid, which is then metabolized to prostaglandins and 15-hydroxyeicosatetraenoic acid (15-HETE). 15-HETE has been shown to produce a significant increase in protein degradation and nuclear binding of the transcription factor NF-κB (a nuclear factor that binds the kappa immunoglobulin light chain gene enhancer in B cells).
Regulation of Protein Synthesis Via Translation Initiation
The role of PIF in the inhibition of protein synthesis is hypothesized to be due to PIF's theorized ability to block translation via RNA-dependent protein kinase (PKR) activation of downstream factors. Inhibition of protein synthesis by PIF is attenuated by insulin at physiological concentrations and below. This suggests that PIF may inhibit protein synthesis at the initiation stage of translation, since insulin regulates protein synthesis through activation of the messenger RNA (mRNA) binding steps in translation initiation.
There are two steps in the initiation of translation that are subject to regulation: (1) the binding of initiator methionyl-transfer RNA (met-tRNA) to the 40s ribosomal subunit; and (2) the binding of mRNA to the 43s preinitiation complex.
In the first step, met-tRNA binds to the 40s ribosomal subunit as a ternary complex with eukaryotic initiation factor 2 (eIF2) and guanosine triphosphate (GTP). Subsequently, the GTP bound to eIF2 is hydrolyzed to guanosine diphosphate (GDP) and eIF2 is released from the ribosomal subunit in a GDP-eIF2 complex. The eIF2 must then exchange the GDP for GTP to participate in another round of initiation. This occurs through the action of another eukaryotic initiation factor, eIF2B, which mediates guanine nucleotide exchange on eIF2. eIF2B is regulated by the phosphorylation of eIF2 on its alpha subunit, which converts it from a substrate into a competitive inhibitor of eIF2B.
In the second step, the binding of mRNA to the 43s preinitiation complex requires a group of proteins collectively referred to as eIF4F, a multisubunit complex consisting of eIF4A (an RNA helicase), eIF4B (which functions in conjunction with eIF4A to unwind secondary structure in the 5′ untranslated region of the mRNA), eIF4E (which binds the m7GTP cap present at the 5′ end of the mRNA), and eIF4G (which functions as a scaffold for eIF4E, eIF4A, and the mRNA). Collectively, the eIF4F complex serves to recognize, unfold, and guide the mRNA to the 43s preinitiation complex. The availability of the eIF4E for the eIF4F complex formation appears to be regulated by the translational repressor eIF4E-binding protein 1 (4E-BP1). 4E-BP1 competes with eIF4G to bind eIF4E and is able to sequester eIF4E into an inactive complex. The binding of 4E-BP1 is regulated through phosphorylation by the kinase mammalian target of rapamycin (mTOR), where increased phosphorylation causes a decrease in the affinity of 4E-BP1 for eIF4E.
It is believed that mTOR is activated by phosphorylation and inhibition of the tuberous sclerosis complex (TSC) 1-TSC2 complex via signaling through the phosphatidylinositol 3 kinase (PI3K)/serine/threonine kinase pathway (PI3K/AKT pathway). mTOR also phosphorylates p70S6 kinase, which phosphorylates ribosomal protein S6, which is believed to enhance the translation of mRNA with an uninterrupted string of pyrimidine residues adjacent to the 5′ cap structure. Proteins encoded by such mRNA include ribosomal proteins, translation elongation factors, and poly-A binding proteins.
Anabolic Factors Involved in Translation Initiation
Many studies have shown that anabolic factors, such as insulin, insulin-like growth factors (IGFs), and amino acids increase protein synthesis and cause muscle hypertrophy. Branched chain amino acids (BCAAs), particularly leucine, can initiate signal transduction pathways that modulate translation initiation. Such pathways often include mTOR. Other studies have demonstrated that mitogenic stimuli, such as insulin and BCAAs, signal via eIF2. As such, amino acid starvation results in an increased phosphorylation of eIF2-α and a decrease in protein synthesis.
Signaling Pathways Involved in Protein Synthesis and Degradation
As noted above, PIF is known to induce protein degradation via the NF-κB pathway. Therefore, it is plausible that inhibition of protein synthesis by PIF occurs via a common signaling initiation point, which then diverges into two separate pathways, one promoting protein degradation via NF-κB and the other inhibiting protein synthesis through mTOR and/or eIF2.
AKT is a serine/threonine kinase, also known as protein kinase B (PKB). Activation of AKT occurs through direct binding of the inositol lipid products of the PI3K to its pleckstrin homology domain. PI3K-dependent activation of AKT also occurs through phosphoinositide-dependent kinase (PDK1)-mediated phosphorylation of threonine 308, which leads to autophosphorylation of serine 473. Although initially believed to operate as components of distinct signaling pathways, several studies have demonstrated that the NF-κB and AKT signaling pathways converge. Studies have shown that AKT signaling inhibits apoptosis in a variety of cell types in vitro, mediated by its ability to phosphorylate apoptosis-regulating components, including IκK, the kinase involved in NF-κB activation. Thus, activation of AKT stimulates activation of NF-κB. Although this would place AKT upstream of NF-κB activation in the sequence of signaling events, one study reports that AKT may be a downstream target of NF-κB. Overall, this suggests that AKT is involved in a catabolic pathway. Other data, however, suggest that AKT is also involved in anabolic processes through activation of mTOR and the consequent phosphorylation of p70S6 kinase and 4E-BP1, leading to an increase in protein synthesis.
PKR is an interferon-induced, RNA-dependent serine/threonine protein kinase responsible for control of an antiviral defense pathway. PKR may be induced by forms of cellular stress other than interferon. Some evidence suggests that tumor necrosis factor (TNF)-alpha also acts through PKR. Interestingly, both interferon and TNF-alpha have been implicated as causative factors of cachectic states. Following interaction with activating stimuli (e.g., insulin, IGF, BCAAs), PKR has been reported to form homodimers and autophosphorylate. As a result, PKR is able to catalyze the phosphorylation of target substrates, the most well-characterised being the phosphorylation of Serine 51 on the eIF2-α subunit. The eIF2 then sequesters eIF2B, a rate-limiting component of translation, resulting in the inhibition of protein synthesis. Recent studies suggest that PKR physically associates with the IκK complex and stimulates NF-κB-inducing kinase (NIK) while phosphorylating IκK, resulting in its subsequent degradation. Some studies suggest that NF-κB is activated by PKR by a mechanism independent from its eIF2 kinase activity, while other studies indicate that the phosphorylation of eIF2-α is required for the activation of NF-κB.
PKR-like ER-resident kinase (PERK) is another kinase that phosphorylates eIF2-α and activates NF-κB. However, it is unlikely that PIF acts through this pathway, since PERK causes the release of IκK from NF-κB, but not its degradation. In addition, PIF has been shown to cause the degradation of IκK during the activation of NF-κB.
Known Treatments for Muscle Loss
Treatment of conditions such as cachexia often includes nutritional supplementation, and, in particular, amino acid supplementation, in an attempt to increase protein synthesis. The three BCAAs are valine, leucine, and isoleucine. Previously, leucine has been shown to function, not only as a protein building block, but also as an inducer of signal transduction pathways that modulate translation initiation. Our recent novel research suggests that all three of the BCAAs possess the ability to reduce protein degradation and enhance protein translation comparably.
Cachexia is just one of the conditions, disorders, and diseases for which amino acid supplementation has proved beneficial. Amino acid supplementation has also been used to treat diabetes, hypertension, high levels of serum cholesterol and triglycerides, Parkinson's disease, insomnia, drug and alcohol addiction, pain, insomnia, and hypoglycemia. Supplementation with BCAAs, in particular, has been used to treat liver disorders, including compromised liver function, including cirrhosis, gall bladder disorders, chorea and dyskinesia, and kidney disorders, including uremia. BCAA supplementation has also proved successful in the treatment of patients undergoing hemodialysis, resulting in improvements in overall health and mood.
To date, the treatment of muscle loss, including treatments involving nutritional supplementation with amino acids, has focused on the promotion of muscle anabolism. For example, U.S. Patent Application Publication No. 2004/0122097 to Verlaan et al. describes nutritional supplements containing both leucine and protein for promoting the generation of muscle tissue. Leucine precursors, such as pyruvate, and metabolites, such as β-hydroxy-β-methylbutyrate and α-ketoisocaproate, exhibit properties similar to those of leucine. Of note, β-hydroxy-β-methylbutyrate is not produced by humans in any clinically relevant quantities and therefore must be supplemented.
Others have shown that insulin, an anabolic hormone, is capable of promoting protein synthesis when administered in large doses. Thus, known treatment approaches, while providing some benefit to individuals suffering from muscle loss through increased generation of muscle tissue, do not affect muscle loss itself. That is, known methods of treating muscle loss are directed toward increasing muscle anabolism rather than decreasing muscle catabolism.
The amino acids that comprise skeletal muscle are in a constant state of flux where new amino acids, either coming from administration by enteral or parenteral routes or recirculated, are deposited as protein and current proteins are degraded. Loss of muscle mass can be the result of many factors including decreased rate of protein synthesis with normal degradation, increased degradation with normal synthesis or an exacerbation of both reduced synthesis and increased degradation. As a result, therapies aimed at increasing synthesis only address one-half of the problem in muscle wasting disease(s).
Accordingly, there is a need in the art for a method of treating muscle loss that decreases muscle catabolism and, optionally, increases muscle anabolism.